Method for inhibiting uterine fibroid disease with 1,1,2-triphenylbut-1-ene derivatives

ABSTRACT

The present invention provides novel methods of inhibiting uterine fibroid disease and endometriosis in women comprising administering to a woman in need of treatment an effective amount of a compound of formula I ##STR1## wherein R 1  and R 2  may be the same or different provided that, when R 1  and R 2  are the same, each is a methyl or ethyl group, and, when R 1  and R 2  are different, one of them is a methyl or ethyl group and the other is a benzyl group, or a pharmaceutically acceptable salt thereof.

This application is a division of application Ser. No. 08/241,258, filedMay 11, 1994 now U.S. Pat. No. 5,455,275.

BACKGROUND OF THE INVENTION

The present invention relates to the discovery that a group of1,1,2-triphenylbut-1-ene derivatives are useful for inhibitingendometriosis and uterine fibroid disease in women.

Unterine fibroid disease (uterine fibrosis) is an old and ever presentclinical problem which goes under a variety of names, including uterinehypertrophy, uterine lieomyomata, myometrial hypertrophy, fibrosisuteri, and fibrotic metritis. Essentially, uterine fibroid disease is acondition where there is an inappropriate deposition of fibroid tissueon the wall of the uterus.

This condition is a cause of dysmenorrhea and infertility in women. Theexact cause of this condition is poorly understood but evidence suggeststhat it is an inappropriate response of fibroid tissue to estrogen. Sucha condition has been produced in rabbits by daily administrations ofestrogen for 3 months. In guinea pigs, the condition has been producedby daily administration of estrogen for four months. Further, in rats,estrogen causes similar hypertrophy.

The most common treatment of uterine fibroid disease involves surgicalprocedures which are both costly and sometimes a source of complicationssuch as the formation of abdominal adhesions and infections. In somepatients, initial surgery is only a temporary treatment and the fibroidsregrow. In those cases, a hysterectmomy is performed which effectivelyends the fibroids, but also the reproductive life of the patient. Also,gonadotropin releasing hormone antagonists may be administered, buttheir use is tempered by the fact they can lead to osteoporosis.

Endometriosis is a condition of severe dysmenorrhea, which isaccompanied by severe pain, bleeding into the endometrial masses orperitoneal cavity, and often leads to infertility. The cause of thesymptoms of this condition appear to be ectopic endometrial growthswhich respond inappropriately to normal hormonal control and are locatedin inappropriate tissues. Because of the inappropriate locations forendometrial growth, the tissue seems to initiate local inflammatory-likeresponses causing macrophage infiltration and a cascade of eventsleading to initiation of the painful response. The exact etiology ofthis disease is not well understood and its treatment by hormonaltherapy is diverse, poorly defined, and marked by numerous unwanted andperhaps dangerous side effects.

One of the treatments for this disease is the use of low dose estrogento suppress endometrial growth through a negative feedback effect oncentral gonadotropin release, and subsequent ovarian production ofestrogen. However, it is sometimes necessary to use continuous estrogento control the symptoms. This use of estrogen can often lead toundesirable side effects and even the risk of endometrial cancer.

Another treatment consists of continuous administration of progestinwhich induces amenorrhea and, by suppressing ovarian estrogenproduction, can cause regressions of the endometrial growths. The use ofchronic progestin therapy is often accompanied by the unpleasant centralnervous system side effects of progestin, and often leads to infertilitydue to suppression of ovarian function.

A third treatment consists of the administration of weak androgens,which are effective in controlling the endometriosis. However, theyinduce severe masculinizing effects. Several of these treatments havealso been implicated in causing a mild degree of bone loss withcontinued therapy.

Therefore, new methods of treating endometriosis are desirable.

SUMMARY OF THE INVENTION

The present invention relates to methods for inhibiting endometriosisand uterine fibroid disease comprising administering to a woman in needof treatment an effective amount of a compound of formula I ##STR2##wherein R¹ and R² may be the same or different provided that, when R¹and R² are the same, each is a methyl or ethyl group, and, when R¹ andR² are different, one of them is a methyl or ethyl group and the otheris a benzyl group, or a pharmaceutically acceptable salt thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns methods for inhibiting uterine fibroiddisease and endometriosis in women. The term "inhibit" is defined toinclude its generally accepted meaning which includes prophylacticallytreating a subject from incurring one or more of these disease states,holding in check the symptoms of such a disease state, and/or treatingsuch symptoms. Thus, the present methods include both medicaltherapeutic and/or prophylactic treatment, as appropriate.

The methods of this invention are practiced by administering to a womanin need of treatment an effective amount of a compound of formula I##STR3## wherein

R¹ and R² may be the same or different provided that, when R¹ and R² arethe same, each is a methyl or ethyl group, and, when R¹ and R² aredifferent, one of them is a methyl or ethyl group and the other is abenzyl group; or a pharmaceutically acceptable salt thereof.

Compounds of formula I are known in the art and essentially are preparedvia the methods described in U.S. Pat. No. 5,047,431 which is hereinincorporated by reference.

A preferred formula I compound is that in which R¹ and R² each aremethyl. This preferred compound is known as droloxifene which previouslyhas been described as an antiestrogenic agent and is useful for thetreatment of hormone-dependent mammary tumors (U.S. Pat. No. 5,047,431)and for the relief of bone diseases caused by the deficiency of estrogenor the like (U.S. Pat. No. 5,254,594). Furthermore, droloxifene is knownto have less of a uterotrophic effect than other antiestrogeniccompounds such as tamoxifen.

Although the free-base form of formula I compounds can be used in themethods of the present invention, it is preferred to prepare and use apharmaceutically acceptable salt form. Thus, the compounds used in themethods of this invention form pharmaceutically acceptable acid and baseaddition salts with a wide variety of inorganic and, preferrably,organic acids and bases, and include the physiologically acceptablesalts which are often used in pharmaceutical chemistry. Such salts arealso part of this invention. Typical inorganic acids used to form suchsalts include hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric,phosphoric, hypophosphoric, and the like. Salts derived from organicacids, such as aliphatic mono and dicarboxylic acids, phenyl substitutedalkanoic acids, hydroxyalkanoic and hydroxyalkandioic acids, aromaticacids, aliphatic and aromatic sulfonic acids, may also be used. Suchpharmaceutically acceptable salts thus include acetate, phenylacetate,trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate,o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate,phenylbutyrate, β-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate,caprate, caprylate, chloride, cinnamate, citrate, formate, fumarate,glycollate, heptanoate, hippurate, lactate, malate, maleate,hydroxymaleate, malonate, mandelate, mesylate, nicotinate,isonicotinate, nitrate, oxalate, phthalate, terephthalate, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, propiolate, propionate, phenylpropionate, salicylate,sebacate, succinate, suberate, sulfate, bisulfate, pyrosulfate, sulfite,bisulfite, sulfonate, benzenesulfonate, p-bromophenylsulfonate,chlorobenzenesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate,methanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,p-toluenesulfonate, xylenesulfonate, tartarate, and the like. Apreferred salt is the citrate salt.

The pharmaceutically acceptable acid addition salts are typically formedby reacting a compound of formula I with an equimolar or excess amountof acid. The reactants are generally combined in a mutual solvent suchas diethyl ether or benzene. The salt normally precipitates out ofsolution within about one hour to 10 days and can be isolated byfiltration or the solvent can be stripped off by conventional means.

The pharmaceutically acceptable salts of formula I compounds generallyhave enhanced solubility characteristics compared to the compound fromwhich they are derived, and thus are often more amenable to formulationas liquids or emulsions.

Once prepared, the free base or salt form of formula I compounds can beadministered to an individual in need of treatment for the methodsherein described. The following non-limiting test examples illustratethe methods of the present invention.

Test Procedure General Preparation Procedure

In the examples illustrating the methods, a post-menopausal model isused to determine the effect of different treatments upon test animaluteri.

Seventy-five day old female Sprague Dawley rats (weight range of 200 to250 g) are obtained from Charles River Laboratories (Portage, Mich.).The animals are either bilaterally ovariectomized (OVX) or exposed to aSham surgical procedure at Charles River Laboratories, and then shippedafter one week. Upon arrival, they are housed in metal hanging cages ingroups of 3 or 4 per cage and have ad libitum access to food (calciumcontent approximately 0.5%) and water for one week. Room temperature ismaintained at 22.2°±1.7° C. with a minimum relative humidity off 40%.The photoperiod in the room is 12 hours light and 12 hours dark.

Dosing Regimen Tissue Collection.

After a one week acclimation period (therefore, two weeks post-OVX)daily dosing with test compound is initiated. 17α-ethynyl estradiol andthe test compound are given orally, unless otherwise stated, as asuspension in 20% cyclodextrin. Animals are dosed daily for 4 days.Following the dosing regimen, animals are weighed and anesthetized witha ketamine: Xylazine (2:1, V:V) mixture and a blood sample is collectedby cardiac puncture. The animals are then sacrificed by asphyxiationwith CO₂, the uterus is removed through a midline incision, and a wetuterine weight is determined.

Uterine Fibrosis Test Procedures

Test 1

Between 3 and 20 women having uterine fibrosis are administered acompound of the present invention. The amount of compound administeredis from 0.1 to 1000 mg/day, and the period of administration is 3months.

The women are observed during the period of administration, and up to 3months after discontinuance of administration, for effects on uterinefibrosis.

Test 2

The same procedure is used as in Test 1, except the period ofadministration is 6 months.

Test 3

The same procedure is used as in Test 1, except the period ofadministration is 1 year.

Test 4

A. Induction of fibroid tumors in guinea pig.

Prolonged estrogen stimulation is used to induce leiomyomata in sexuallymature female guinea pigs. Animals are dosed with estradiol 3-5 timesper week by injection for 2-4 months or until tumors arise. Treatmentsconsisting of a compound of the invention or vehicle is administereddaily for 3-16 weeks and then animals are sacrificed and the uteriharvested and analyzed for tumor regression.

B. Implantation of human uterine fibroid tissue in nude mice.

Tissue from human leiomyomas are implanted into the peritoneal cavityand or uterine myometrium of sexually mature, castrated, female, nudemice. Exogenous estrogen are supplied to induce growth of the explantedtissue. In some cases, the harvested tumor cells are cultured in vitroprior to implantation. Treatment consisting of a compound of the presentinvention or vehicle is supplied by gastric lavage on a daily basis for3-16 weeks and implants are removed and measured for growth orregression. At the mime of sacrifice, the uteri is harvested to assessthe status of the organ.

Test 5

A. Tissue from human uterine fibroid tumors is harvested and maintained,in vitro, as primary nontransformed cultures. Surgical specimens arepushed through a sterile mesh or sieve, or alternately teased apart fromsurrounding tissue to produce a single cell suspension. Cells aremaintained in media containing 10% serum and antibiotic. Rates of growthin the presence and absence of estrogen are determined. Cells areassayed for their ability to produce complement component C3 and theirresponse co growth factors and growth hormone. In vitro cultures areassessed for their proliferative response following treatment withprogestins, GnRH, a compound of the present invention and vehicle.Levels of steroid hormone receptors are assessed weekly to determinewhether important cell characteristics are maintained in vitro. Tissuefrom 5-25 patients are utilized.

Activity in at least one of the above tests indicates the compounds ofthe present invention are of potential in the treatment of uterinefibrosis.

Endometriosis Test Procedure

In Tests 1 and 2, effects of 14-day and 21-day administration ofcompounds of the present invention on the growth of explantedendometrial tissue can be examined.

Test 1

Twelve to thirty adult CD strain female rats are used as test animals.They are divided into three groups of equal numbers. The estrous cycleof all animals is monitored. On the day of proestrus, surgery isperformed on each female. Females in each group have the left uterinehorn removed, sectioned into small squares, and the squares are looselysutured at various sites adjacent to the mesenteric blood flow. Inaddition, females in Group 2 have the ovaries removed.

On the day following surgery, animals in Groups 1 and 2 receiveintraperitoneal injections on water or 14 days whereas animals in Group3 receive intraperitoneal injections of 1.0 mg of a compound of thepresent invention per kilogram of body weight for the same duration.Following 14 days of treatment, each female is sacrificed and theendometrial explants, adrenals, remaining uterus, and ovaries, whereapplicable, are removed and prepared for histological examination. Theovaries and adrenals are weighed.

Test 2

Twelve to thirty adult CD strain female rats are used as test animals.They are divided into two equal groups. The estrous cycle of all animalsis monitored. On the day of proestrus, surgery is performed on eachfemale. Females in each group have the left uterine horn removed,sectioned into small squares, and the squares are loosely sutured atvarious sites adjacent to the mesenteric blood flow.

Approximately 50 days following surgery, animals assigned to Group 1receive intraperitoneal injections of water for 21 days whereas animalsin Group 2 receive intraperitoneal injections of 1.0 mg of a compound ofthe present invention per kilogram of body weight for the same duration.Following 21 days of treatment, each female is sacrificed and theendometrial explants and adrenals are removed and weighed. The explantsare measured as an indication of growth. Estrous cycles are monitored.

Test 3

A. Surgical induction of endometriosis

Autographs of endometrial tissue are used to induce endometriosis inrats and/or rabbits. Female animals at reproductive maturity undergobilateral oophorectomy, and estrogen is supplied exogenously thusproviding a specific and constant level of hormone. Autologousendometrial tissue is implanted in the peritoneum of 5-150 animals andestrogen supplied to induce growth of the explanted tissue. Treatmentconsisting of a compound of the present invention is supplied by gastriclavage on a daily basis for 3-16 weeks, and implants are removed andmeasured for growth or regression. At the time of sacrifice, the intacthorn of the uterus is harvested to assess status of endometrium.

B. Implantation of human endometrial tissue in nude mice.

Tissue from human endometrial lesions is implanted into the peritoneumof sexually mature, castrated, female, nude mice. Exogenous estrogen issupplied to induce growth of the explanted tissue. In some cases, theharvested endometrial cells are cultured in vitro prior to implantation.Treatment consisting of a compound of the present invention supplied bygastric lavage on a daily basis for 3-16 weeks, and implants are removedand measured for growth or regression. At the time of sacrifice, theuteri is harvested to assess the status of the intact endometrium.

Test 4

A. Tissue from human endometrial lesions is harvested and maintained invitro as primary nontransformed cultures. Surgical specimens are pushedthrough a sterile mesh or sieve, or alternately teased apart fromsurrounding tissue to produce a single cell suspension. Cells aremaintained in media containing 10% serum and antibiotic. Rates of growthin the presence and absence of estrogen are determined. Cells areassayed for their ability to produce complement component C3 and theirresponse to growth factors and growth hormone. In vitro cultures areassessed for their proliferative response following treatment withprogestins, GnRH, a compound of the invention, and vehicle. Levels ofsteroid hormone receptors are assessed weekly to determine whetherimportant cell characteristics are maintained in vitro. Tissue from 5-25patients is utilized.

Activity in any of the above assays indicates that the compounds of thepresent invention are useful in the treatment of endometriosis.

For the methods of the present invention, compounds of Formula I areadministered continuously, from 1 to 4 times daily. However, cyclicaltherapy may especially be useful in the treatment of endometriosis ormay be used acutely during painful attacks of the disease.

As used Herein, the term "effective amount" means an amount of compoundof the methods of the present invention which is capable of inhibitingthe symptoms of the pathological conditions herein described. Thespecific dose of a compound administered according to this inventionwill, of course, be determined by the particular circumstancessurrounding the case including, for example, the compound administered,the route of administration, the state of being of the patient, and theseverity of the pathological condition being treated. A typical dailydose will contain a nontoxic dosage level of from about 0.25 mg no about400 mg/day of a compound of the present invention. Preferred daily dosesgenerally will be from about 1 mg to about 20 mg/day.

The compounds of this invention can be administered by a variety ofroutes including oral, rectal, transdermal, subucutaneus, intravenous,intramuscular, and intranasal. These compounds preferably are formulatedprior to administration, the selection of which will be decided by theattending physician. Typically, a formula I compound, or apharmaceutically acceptable salt thereof, is combined with apharmaceutically acceptable carrier, diluent or excipient to form apharmaceutical formulation.

The total active ingredients in such formulations comprises from 0.1% to99.9% by weight of the formulation. By "pharmaceutically acceptable" itis meant the carrier, diluent, excipients, and/or salt must becompatible with the other ingredients of the formulation, and notdeleterious to the recipient thereof.

Pharmaceutical formulations containing a compound of formula I can beprepared by procedures known in the art using well known and readilyavailable ingredients. For example, the compounds of formula I can beformulated with common excipients, diluents, or carriers, and formedinto tablets, capsules, suspensions, powders, and the like. Examples ofexcipients, diluents, and carriers that are suitable for suchformulations include the following: fillers and extenders such asstarch, sugars, mannitol, and silicic derivatives; binding agents suchas carboxymethyl cellulose and other cellulose derivatives, alginates,gelatin, and polyvinyl-pyrrolidone; moisturizing agents such asglycerol; disintegrating agents such as calcium carbonate and sodiumbicarbonate; agents for retarding dissolution such as paraffin;resorption accelerators such as quaternary ammonium compounds; surfaceactive agents such as cetyl alcohol, glycerol monostearate; adsorptivecarriers such as kaolin and bentonite; and lubricants such as talc,calcium and magnesium stearate, and solid polyethyl glycols.

The compounds also can be formulated as elixirs or solutions forconvenient oral administration or as solutions appropriate forparenteral administration, for example, by intramuscular, subcutaneousor intravenous routes.

Additionally, the compounds are well suited to formulation as sustainedrelease dosage forms and the like. The formulations can be soconstituted that they release the active ingredient only or preferablyin a particular physiological location, possibly over a period of time.The coatings, envelopes, and protective matrices may be made, forexample, from polymeric substances or waxes.

Compounds of formula I generally will be administered in a convenientformulation. The following formulation examples only are illustrativeand are not intended to limit the scope of the present invention.

Formulations

In the formulations which follow, "active ingredient" means a compoundof formula I, or a salt thereof.

    ______________________________________                                        Formulation 1: Gelatin Capsules                                               Hard gelatin capsules are prepared using the following:                       Ingredient        Quantity (mg/capsule)                                       ______________________________________                                        Active ingredient 0.25-400                                                    Starch, NF         0-650                                                      Starch flowable powder                                                                          0-50                                                        Silicone fluid 350 centistokes                                                                  0-15                                                        ______________________________________                                    

The formulation above may be changed in compliance with the reasonablevariations provided.

A tablet formulation is prepared using the ingredients below:

    ______________________________________                                        Formulation 2: Tablets                                                        Ingredient       Quantity (mg/tablet)                                         ______________________________________                                        Active ingredient                                                                              0.25-400                                                     Cellulose, microcrystalline                                                                    200-650                                                      Silicon dioxide, fumed                                                                          10-650                                                      Stearate acid     5-15                                                        ______________________________________                                    

The components are blended and compressed to form tablets.

Alternatively, tablets each containing 0.25-400 mg of active ingredientare made up as follows:

    ______________________________________                                        Formulation 3: Tablets                                                        Ingredient          Quantity (mg/tablet)                                      ______________________________________                                        Active ingredient   0.25-400                                                  Starch              45                                                        Cellulose, microcrystalline                                                                       35                                                        Polyvinylpyrrolidone                                                                              4                                                         (as 10% solution in water)                                                    Sodium carboxymethyl cellulose                                                                    4.5                                                       Magnesium stearate  0.5                                                       Talc                1                                                         ______________________________________                                    

The active ingredient, starch, and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders which are thenpassed through a No. 14 mesh U.S. sieve. The granules so produced aredried at 50°-60° C. and passed through a No. 18 mesh U.S. sieve. Thesodium carboxymethyl starch, magnesium stearate, and talc, previouslypassed through a No. 60 U.S. sieve, are then added to the granuleswhich, after mixing, are compressed on a tablet machine to yieldtablets.

Suspensions each containing 0.25-400 mg of medicament per 5 ml dose aremade as follows:

    ______________________________________                                        Formulation 4: Suspensions                                                    Ingredient           Quantity (mg/5 ml)                                       ______________________________________                                        Active ingredient    0.25-400  mg                                             Sodium carboxymethyl cellulose                                                                     50        mg                                             Syrup                1.25      mg                                             Benzoic acid solution                                                                              0.10      mL                                             Flavor               q.v.                                                     Color                q.v.                                                     Purified water to    5         mL                                             ______________________________________                                    

The medicament is passed through a No. 45 mesh U.S. sieve and mixed withthe sodium carboxymethyl cellulose and syrup to form a smooth paste. Thebenzoic acid solution, flavor, and color are diluted with some of thewater and added, with stirring. Sufficient water is then added toproduce the required volume.

An aerosol solution is prepared containing the following ingredients:

    ______________________________________                                        Formulation 5: Aerosol                                                        Ingredient           Quantity (% by weight)                                   ______________________________________                                        Active ingredient    0.25                                                     Ethanol              25.75                                                    Propellant 22 (Chlorodifluoromethane)                                                              70.00                                                    ______________________________________                                    

The active ingredient is mixed with ethanol and the mixture added to aportion of the propellant 22, cooled to 30° C., and transferred to afilling device. The required amount is then fed to a stainless steelcontainer and diluted with the remaining propellant. The valve units arethen fitted to the container.

Suppositories are prepared as follows:

    ______________________________________                                        Formulation 6: Suppositories                                                  Ingredient       Quantity (mg/suppository)                                    ______________________________________                                        Active ingredient                                                                              250                                                          Saturated fatty acid glycerides                                                                2,000                                                        ______________________________________                                    

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimal necessary heat. The mixture is then poured into asuppository mold of nominal 2 g capacity and allowed to cool.

An intravenous formulation is prepared as follows:

    ______________________________________                                        Formulation 7: Intravenous Solution                                           Ingredient            Quantity                                                ______________________________________                                        Active ingredient     20     mg                                               Isotonic saline       1,000  mL                                               ______________________________________                                    

The solution of the above ingredients is intravenously administered to apatient at a rate of about 1 mL per minute.

I claim:
 1. A method for inhibiting uterine fibroid disease comprisingadministering to a woman in need thereof an effective amount of acompound of formula I ##STR4## wherein R¹ and R² may be the same ordifferent provided that, when R¹ and R² are the same, each is a methylor ethyl group, and, when R¹ and R² are different, one of them is amethyl or ethyl group and the other is a benzyl group; or apharmaceutically acceptable salt thereof.
 2. A method according to claim1 wherein the compound of formula I is a compound wherein R¹ and R² eachare methyl, or a pharmaceutically acceptable salt thereof.
 3. A methodaccording to claim 2 wherein said salt thereof is the citrate salt.